Molecular beacon polymerase chain reaction detection of Escherichia coli O157 : H7 in milk

A fluorescently labeled oligonucleotide probe (molecular beacon) was applie d to detect Escherichia coil O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the sit-II gene coding fo r the A subunit and to fluorescence when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificia lly contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coil O157:H7 per mi and was found to correlate with the amount of template in each reaction. F luorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated posit ive results more rapidly than traditional agarose gel electrophoresis analy sis of PCR products. Use of molecular beacons allows real-time monitoring o f PCR reactions, and the closed-tube format allows simultaneous detection a nd confirmation of target amplicons without the need for agarose gel electr ophoresis and/or Southern blotting. This is the first report of a stem-and- loop molecular beacon being applied for direct detection of a pathogen in f ood.